CN116615261A - Crosslinked hyaluronic acid hydrogel crosslinked using crosslinking agent and polyol and filler comprising same - Google Patents

Abstract

The object of the present invention is to provide a cross-linked hyaluronic acid hydrogel in which a cross-linking agent and a polyol are used to reduce toxicity during cross-linking, thereby enhancing safety while improving durability in vivo, and the present invention also Filling compositions comprising the cross-linked hyaluronic acid hydrogel are provided.

Description

Cross-linked hyaluronic acid hydrogel cross-linked using cross-linking agent and polyol and containing it filler

technical field

The present invention relates to hyaluronic acid hydrogels crosslinked using polyols and crosslinking agents and fillers comprising the same.

Background technique

Fillers developed for wrinkle improvement widely use hyaluronic acid, a natural polymer with high biocompatibility. To increase the duration of hyaluronic acid in the body, hyaluronic acid hydrogels cross-linked using various cross-linking agents have been used as fillers. However, since the crosslinking agent generally has high toxicity, there is a disadvantage that it is difficult to use in a large amount. In addition, when the cross-linking rate is low despite the formation of a cross-linked hydrogel, there is a disadvantage of being rapidly decomposed by hyaluronidase or reactive oxygen species (free radicals), so it is difficult to improve both stability and durability in vivo. By.

Contents of the invention

technical problem

As a result of repeated studies to solve the problems of the prior art as described above, the present inventors confirmed that when hyaluronic acid is crosslinked using a polyhydric alcohol such as sugar alcohol together with a conventionally used crosslinking agent, excellent improvement Stability against hyaluronidase, reactive oxygen species (free radicals) and/or heat, and significantly reduced toxicity due to the low amount of cross-linking used allows the preparation of highly biocompatible fillers, thereby Complete the present invention.

Accordingly, an object of the present invention is to provide a cross-linked hyaluronic acid hydrogel with reduced toxicity during cross-linking to have high safety and high durability in vivo, and a filler comprising it.

technical solution

According to one aspect of the present invention, a cross-linked hyaluronic acid hydrogel is provided, the cross-linked hyaluronic acid hydrogel comprises hyaluronic acid or a salt thereof, a cross-linking agent and a polyhydric alcohol, wherein the hyaluronic acid An acid or a salt thereof is cross-linked with the cross-linking agent and polyol, and a filler comprising the cross-linked hyaluronic acid hydrogel is also provided.

According to one aspect of the present invention, the filler is used for soft tissue injection, such as for skin injection, and the filler can be used for filling properties, such as filler use, such as filling of biological tissue, wrinkle improvement for filling wrinkles , facial reshaping or contour correction or soft tissue volume restoration or augmentation.

According to one aspect of the present invention, the polyol may be a sugar alcohol.

According to one aspect of the present invention, a method for preparing the cross-linked hyaluronic acid hydrogel can be provided, the method comprising mixing hyaluronic acid or its salt with a polyhydric alcohol, and mixing a cross-linking agent and an aqueous alkali solution The mixed solution is added to the mixture of hyaluronic acid or its salt and polyol and allowed to react.

According to one aspect of the present invention, there is provided a prefilled syringe filled with a filler comprising the cross-linked hyaluronic acid.

beneficial effect

Due to the use of cross-linking agents and polyols in the cross-linked hyaluronic acid hydrogel according to the present invention, the resistance to hyaluronidases in vivo, reactive oxygen species and storage temperature is increased. Therefore, fillers comprising this cross-linked hyaluronic acid hydrogel have improved stability and reduced toxicity during storage, thereby exhibiting high safety and excellent persistence in vivo, thus making soft tissue repair or volume augmentation possible. Great wrinkle improvement effect.

Description of drawings

1 is a graph comparing the results of in vitro enzyme resistance of cross-linked hyaluronic acid hydrogel cross-linked using a cross-linking agent and mannitol according to Example 1-1 of the present invention and Comparative Examples 2 to 4. FIG.

2 is a graph comparing the results of in vitro free radical resistance of the cross-linked hyaluronic acid hydrogel cross-linked using a cross-linking agent and mannitol according to Example 1-1 of the present invention and Comparative Examples 2 to 4. FIG.

3 is a graph comparing the results of in vitro heat resistance of the cross-linked hyaluronic acid hydrogel cross-linked using a cross-linking agent and mannitol according to Example 1-1 of the present invention and Comparative Examples 2 to 4. FIG.

4 is a graph comparing the in vivo biocompatibility test results of the cross-linked hyaluronic acid hydrogel cross-linked using a cross-linking agent and mannitol according to Example 1-1 of the present invention and Comparative Examples 2 to 4. FIG.

Detailed ways

Hereinafter, the present invention will be described in detail.

Hyaluronic acid (hereinafter also referred to as "HA") contained in the cross-linked hyaluronic acid hydrogel of the present invention is a biopolymer substance composed of N-acetyl-D-glucosamine and D- The disaccharide repeating unit (disaccharide unit) composed of glucuronic acid is linearly linked, abundantly present in the vitreous humor of the eye, synovial fluid of the joint, cockscomb, etc., and has excellent biocompatibility, so it is widely used For medical treatment and medical devices or cosmetic purposes, such as ophthalmic surgery aids, joint viscosity supplements, drug delivery materials, eye drops, wrinkle improvers, etc.

Specifically, the hyaluronic acid contained in the hyaluronic acid hydrogel according to the present invention may refer to hyaluronic acid or a salt of hyaluronic acid. Salts of hyaluronic acid include, for example, all organic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate and tetrabutylammonium hyaluronate, but Not limited to this.

In the present invention, the weight average molecular weight of the hyaluronic acid used for the crosslinking reaction may be 1,000,000 Da or more, 1,500,000 Da or more, 2,000,000 Da or more, 2,300,000 Da or more, or 2,500,000 Da or more, For example it is 1,000,000 to 1,500,000Da, 1,000,000 to 2,000,000Da, 1,000,000 to 3,000,000Da, 1,000,000 to 4,000,000Da, 1,500,000 to 2,000,000Da, 1,500,000 to 3,000 0,000Da, 1,500,000 to 4,000,000Da, 2,000,000 to 4,000,000Da, 2,300,000 to 4,000,000Da, 2,000,000 to 3,700,000 Da, 2,200,000 to 3,700,000 Da, or 2,500,000 to 3,500,000 Da.

The cross-linked hyaluronic acid hydrogel according to the present invention is characterized in that the hyaluronic acid or its salt is cross-linked using a cross-linking agent and a polyhydric alcohol.

The term "cross-linking" as used in the present invention refers to intermolecular bonds that combine individual polymer molecules or monomer chains into a more stable structure such as a gel. Thus, a crosslinked polymer has at least one intermolecular bond linking at least one individual polymer molecule to another polymer molecule.

The cross-linked hyaluronic acid hydrogel according to the present invention is characterized in that the hyaluronic acid or its salt is cross-linked using a cross-linking agent and a polyol, unlike conventional hyaluronic acid hydrogels cross-linked only with a cross-linking agent. glue.

The term "crosslinking agent" refers to any compound capable of inducing crosslinking between hyaluronic acid chains, and a crosslinking agent that can crosslink hyaluronic acid or a salt thereof can be used without limitation in the present invention, and for example It is variable, for example as a compound comprising two or more epoxy functional groups. As preferred examples of the crosslinking agent, endogenous polyamines, aldehydes, carbodiimides, divinylsulfone may be included as non-epoxy crosslinking agents. In addition, the epoxy crosslinking agent may include butanediol diglycidyl ether (1,4-butanediol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether (1,6-Hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol Polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, bisglycidoxyethylene (1,2-(bis(2,3-glycidyloxypropoxy) base) ethylene), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, etc., among which diepoxide-based 1,4-butanediol diglycidyl ether is particularly preferred because of low toxicity.

A polyol that cross-links hyaluronic acid or an acceptable salt thereof together with the cross-linking agent is linked to the cross-linking agent to cross-link hyaluronic acid or an acceptable salt thereof. This polyol refers to an organic molecule containing 2 or more free hydroxyl groups, and it may be a polyol having 2 to 20 carbon atoms, more specifically a sugar alcohol. Polyols suitable for the present invention may be saturated or unsaturated, linear, branched or cyclic alkylated compounds having at least 2 -OH functional groups on the alkyl chain, for example 3 or more -OH functional groups, especially 4 or more -OH functional groups. Non-limiting examples of said polyols are as follows: glycerol, 1,3-propanediol, isopentyl glycol, pentylene glycol, hexylene glycol; glycols such as ethylene glycol, propylene glycol, butylene glycol, diethylene glycol; Polyglycerols of 2 to 6 repeating units such as diglycerol; erythritol, arabitol, calendula, sorbitol, mannitol, xylitol, dulcitol, glucose, fructose, xylose , trehalose, maltose, sucrose, lactose; and their derivatives and mixtures, such as phosphomethylglucoside. More specifically, the polyol may be selected from mannitol, sorbitol and xylitol. In the present invention, in the polyol, the —OH functional group reacts with an active oxygen species to deactivate the active oxygen species, thereby suppressing decomposition of the filler by the active oxygen species.

As a specific example, the hyaluronic acid or an acceptable salt thereof can be linked with a cross-linking agent and a polyol as described below.

In other words, the hydroxyl group of hyaluronic acid or an acceptable salt thereof is linked to one of the epoxide groups at both ends of the crosslinking agent, and the epoxide group at the other end of the crosslinking agent The group is connected with the hydroxyl group of the polyol and becomes the form of hyaluronic acid-crosslinking agent-polyol. In addition, another hydroxyl group of the polyol is continuously connected to a hydroxyl group of a new hyaluronic acid or an acceptable salt thereof as described below, and finally a cross-linked hyaluronic acid hydrogel is prepared.

The cross-linked hyaluronic acid hydrogel according to the present invention has a degree of modification (MoD) ranging from about 3 to about 100, preferably from about 3 to about 80, more preferably from about 3 to about 50.

Furthermore, in the present invention, the term "modification degree (MoD)" refers to the molar ratio of the total cross-linking agent attached to hyaluronic acid to the moles of total unit hyaluronic acid, and it can be expressed as in Equation 1 below .

[equation 1]

Modification degree (MoD) = number of moles of cross-linking agent attached to hyaluronic acid/moles of unit hyaluronic acid

The cross-linked hyaluronic acid hydrogel according to the present invention may comprise from about 10 mg/g to about 40 mg/g, preferably from about 15 mg/g to about 35 mg/g, based on the total weight of the cross-linked hyaluronic acid hydrogel , more preferably from about 20 mg/g to about 30 mg/g total hyaluronic acid.

The cross-linked hyaluronic acid hydrogel according to the present invention exhibits the effect of modifying the decomposition of hyaluronic acid by active oxygen species, wherein polyols are chemically bound to hyaluronic acid or its salts, and structurally interfere with the activity of hyaluronidase Mechanism of binding and disintegration, so it has the advantage of exhibiting the effect of inhibiting the disintegration of the filler comprising the cross-linked hyaluronic acid hydrogel when injected into the human body, resulting in increased persistence after injection into the body . In addition, when the β-1-4-glycosidic bond where the chemical bond between hyaluronic acid molecules is relatively weak is broken not only by the enzyme as described above but also by heat, the cross-linked hyaluronic acid hydrogel may decompose, Whereas, according to the present invention, the polyol has thermal stability to inhibit thermal decomposition of such hyaluronic acid, and is advantageous during storage, so it is very useful as a filler.

As another aspect, the present invention relates to a method for preparing the cross-linked hyaluronic acid hydrogel using a cross-linking agent and a polyol. Specifically, the preparation method of the cross-linked hyaluronic acid hydrogel according to the present invention may include: (i) mixing hyaluronic acid or its salt with polyhydric alcohol; and (ii) mixing the cross-linking agent and alkali aqueous solution The solution is added to the mixture of hyaluronic acid or its salt and polyol and allowed to react.

In the production method, as for matters related to hyaluronic acid or a salt thereof, a cross-linking agent, and a polyol, unless otherwise stated, matters related to the cross-linked hyaluronic acid hydrogel can be the same applicable.

Furthermore, the weight average molecular weight of the hyaluronic acid or its salt may be 1,000,000 Da or more, 1,500,000 Da or more, 2,000,000 Da or more, 2,300,000 Da or more or 2,500,000 Da or more, for example, it is 1,000,000 to 1,500,000Da, 1,000,000 to 2,000,000Da, 1,000,000 to 3,000,000Da, 1,000,000 to 4,000,000Da, 1,500,000 to 2,000,000Da, 1,500,000 to 3,000Da, 000Da, 1,500,000 to 4,000,000Da, 2,000,000 to 4,000,000Da, 2,300,000 to 4,000,000Da, 2,000,000 to 3,700,000Da, 2,200,000 to 3,700,000 Da or 2,500,000 to 3,500,000 Da.

On the other hand, the concentration of the polyhydric alcohol used in the preparation method may be 5 to 100 mol%, 10 to 100 mol%, 10 to 90 mol%, or 10 to 80 mol% based on hyaluronic acid or its salt, but not limited to This, and can be adjusted appropriately according to the reaction conditions.

In addition, the aqueous alkali solution can be used without limitation as long as it is an aqueous alkali solution known to be suitable for crosslinking of hyaluronic acid, for example, it may be NaOH, KOH, NaHCO ₃ , LiOH or a combination thereof, and Preferably it may be NaOH. The concentration of the alkali aqueous solution may be 0.1 to 0.5N, but is not limited thereto. The concentration of the aqueous alkali solution used in the preparation method may be 5 to 100 mol%, 10 to 100 mol%, 10 to 90 mol%, or 10 to 80 mol% based on hyaluronic acid or a salt thereof, but not limited thereto, and may Adjust appropriately according to the reaction conditions.

When the concentration of the crosslinking agent and/or polyol is used at a high concentration exceeding the above range, a filler having too high elasticity is obtained, while when the concentration is lower than the above range, the elasticity is too low to exhibit Appropriate viscoelasticity. Specifically, the crosslinking reaction can be performed by stirring a mixture of hyaluronic acid or a salt thereof and a polyhydric alcohol, a crosslinking agent, and an aqueous alkali solution to uniformly mix them, and then maintaining them for a certain period of time. The temperature during the crosslinking reaction may be room temperature or higher, preferably in the temperature range of 25 to 65°C or 27 to 55°C, for 1 to 22 hours or 2 to 20 hours.

In the preparation method according to the present invention, the total reaction concentration (ie the ratio of the sum of the weight of hyaluronic acid and polyol to the total weight of hyaluronic acid, polyol and solvent) can be 5 to 30% (w/w) or 10 to 30% (w/w).

In a specific example of the invention, the substance is prepared as follows: 10 mol % of mannitol, sorbitol or xylitol, based on hyaluronic acid, is mixed with hyaluronic acid, mixed with NaOH solution and 1,4-BDDE The solution was mixed and maintained at 30 to 50°C for 2 hours, then the cross-linked gel was washed/neutralized/swelled, then crushed using a sieve, and then sterilized.

In other aspects, the invention relates to a filler comprising said cross-linked hyaluronic acid hydrogel.

The filler may comprise cross-linked hyaluronic acid hydrogel in an amount of 0.5 to 10 wt%, preferably 1 to 5 wt%, based on the total filler weight.

In addition, the filler containing cross-linked hyaluronic acid hydrogel according to the present invention may contain an anesthetic in addition to the cross-linked hyaluronic acid hydrogel to relieve pain of the patient during injection.

The anesthetics include at least one anesthetic known in the art, preferably a local anesthetic, and the concentration of one or more such anesthetics is an amount effective to relieve pain experienced upon injection of the composition. Examples of anesthetics may be selected from ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betocaine Betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, bupivacaine ( butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine ), quinicaine (dimethysoquin), dimethylcaine (dimethocaine), diperodon (diperodon), dicyclomine (dycyclonine), ecgonidine (ecgonine), ecgonine (ecgonine), ethyl chloride, according etidocaine, beta-eucaine, euprocin, phenalcomine, formocaine, hexylcaine, hydroxy Hydroxytetracaine, isobutyl p-aminobenzoate, leucinocainemesylate, levoxadrol, lidocaine, mepivacaine, mepivacaine meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, hydroxy Oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine Propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, methocaine ( trimecaine), zolamine and their salts. In one embodiment, the anesthetic may be lidocaine, for example in the form of lidocaine hydrochloride.

In the filler comprising cross-linked hyaluronic acid hydrogel according to the present invention, the concentration of the anesthetic agent contained in the filler may be about 0.1% by weight to about 1.0% by weight based on the total weight of the filler, For example from about 0.2% to about 0.5% by weight of the composition. Preferably, it may be 0.3% by weight.

The concentration of the anesthetic agent in the filler according to the invention may be therapeutically effective, meaning a concentration suitable to provide advantages in terms of surgical convenience and patient compliance without harming the patient.

In addition, the filler according to the present invention may further contain a buffer solution, and the buffer solution may be used without limitation as long as it is used to prepare hyaluronic acid hydrogel. Examples of preferred buffer solutions may include buffer solutions comprising one or more selected from the group consisting of citric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, diethylbarbituric acid, sodium acetate, TAPS (tri (Hydroxymethyl)methylamino)propanesulfonate), Bicine (2-bis(2-hydroxyethyl)amino)acetate), Tris (tris(hydroxymethyl)ammonium methane), Tricine (N -(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonate), TES (2-[[ 1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]methanesulfonate) and PIPES (piperazine-N,N′-bis(2-ethanesulfonate)), but Not limited to this. The content of the above-mentioned components contained in the buffer solution may be appropriately adjusted, but preferably, it may be contained at a concentration of 0.3 to 2.0 g/L based on the buffer solution.

In addition, the filler according to the present invention may also contain an isotonic agent, and such an isotonic agent may be used without limitation as long as it is used to prepare the filler and it can be contained in the buffer solution. As a preferable isotonic agent, sodium chloride can be used, but not limited thereto. The content of the isotonic agent can be appropriately adjusted if necessary, for example, it can be included at 7.0 to 9.0 g/L based on the buffer solution, but not limited thereto.

In one example according to the present invention, a buffer solution comprising sodium chloride, disodium hydrogen phosphate and sodium dihydrogen phosphate in water for injection is used.

As another aspect, the filler comprising cross-linked hyaluronic acid hydrogel according to the present invention may contain acceptable components included in the preparation of the filler in addition to the above-mentioned components.

Fillers according to the invention comprising cross-linked hyaluronic acid hydrogels have the advantage that the shelf life can be significantly increased compared to conventional hyaluronic acid filler formulations due to their high thermal stability. In addition, it is highly resistant to degradation caused by hyaluronidase and reactive oxygen species (free radicals), thus allowing a significantly increased duration after injection into the human body. Because it exhibits very excellent properties in terms of biocompatibility and toxicity, it can be very useful for cosmetic or therapeutic purposes.

As a particular aspect, fillers comprising cross-linked hyaluronic acid hydrogels according to the present invention can be used for wrinkle improvement by filling biological tissues and filling wrinkles, for facial remodeling or for soft tissues such as lips, nose, buttocks, cheeks Or repair or volume increase of breast etc. The filler comprising hyaluronic acid hydrogel may be administered in an administration form suitable for the purpose, and it may preferably be an injection, more preferably a prefilled injection (prefilled syringe).

As another aspect, the present invention relates to a method for preparing a filler comprising cross-linked hyaluronic acid hydrogel as described above, the method comprising the following steps:

(a) mixing hyaluronic acid or a salt thereof with a polyol;

(b) adding a mixed solution of a crosslinking agent and an aqueous alkali solution to the mixture of hyaluronic acid or its salt and a polyol and reacting it to prepare a crosslinked hyaluronic acid hydrogel;

(c) rough cutting the hyaluronic acid hydrogel prepared in (b);

(d) washing and swelling the roughly cut hyaluronic acid hydrogel prepared in (b) with a buffer solution; and

(e) Pulverize the hyaluronic acid hydrogel washed and swollen in (d).

Steps (a) and (b) are steps of preparing a crosslinked hyaluronic acid hydrogel by crosslinking hyaluronic acid or a salt thereof using a crosslinking agent and a polyol in an aqueous alkali solution, and for the hyaluronic acid with the or its salt, cross-linking agent, polyol and cross-linked hyaluronic acid hydrogel, those mentioned in said cross-linked hyaluronic acid hydrogel and its preparation method are equally applicable.

In the rough cutting process (step (c)), various rough cutting processes of the hyaluronic acid hydrogel can be used. In one example, the cross-linked hyaluronic acid hydrogel prepared after the reaction can be obtained in the shape of a cake (or cylinder), and it can be divided into half-moon shapes, for example, divided into 6 parts using a cutting machine such as a guillotine. Then, a rough cutting process may be performed by passing the gel divided as described above through a cutter with a constant blade interval, preferably two or more times.

The buffer solution used in step (d) can be prepared according to known buffer solution preparation methods. In addition, in the buffer solution, an anesthetic may be further contained additionally. The buffer solution can be used without limitation as long as it is used for the preparation of hyaluronic acid hydrogel. As an example of such a preferred buffer solution, a buffer solution comprising one or more selected from the group consisting of citric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, diethylbarbituric acid, sodium acetate, TAPS (tris(hydroxymethyl)methylamino)propanesulfonate), Bicine (2-bis(2-hydroxyethyl)amino)acetate), Tris (tris(hydroxymethyl)ammonium methane), Tricine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine), HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonate), TES (2- [[1,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]methanesulfonate) and PIPES (piperazine-N,N′-bis(2-ethanesulfonate)) , but not limited to this.

Also, washing and swelling can be repeated once or twice. When washing and swelling are complete, the washing solution can be removed.

Step (e) is a step of pulverizing the washed and swollen hydrogel, and this pulverization can be performed by various pulverization methods, but preferably it is extrusion pulverization.

As another aspect, the cross-linked hydrogel filler prepared after step (e) can be subjected to processes such as sterilization and/or defoaming, and it can be bulk-filled in suitable containers such as pre-filled syringes , sealed and sterilized.

Mode of Carrying Out the Invention

Hereinafter, in order to help understanding of the present invention, a detailed description will be made using Examples. However, the following examples are only intended to illustrate the contents of the present invention, but the scope of the present invention is not limited by the following examples. The embodiments of the present invention are provided in order to more fully explain the present invention to those skilled in the art.

[Example]

Example 1-1: According to the present invention using mannitol and cross-linking agent cross-linked cross-linked hyaluronic acid hydrogel preparation (1)

In order to prepare the cross-linked hyaluronic acid hydrogel cross-linked using mannitol and a cross-linking agent according to the present invention, the following procedure was performed.

Specifically, hyaluronic acid sodium salt with an average molecular weight of 3 million Da, sodium hydroxide, BDDE (1,4-butanediol diglycidyl ether) as a crosslinking agent, and mannitol were weighed out, respectively.

1 g of hyaluronic acid and 10 mol% mannitol based on the weight of hyaluronic acid were weighed out and then placed in a mixer container and mixed. To another container (50 mL tube), 0.25 N sodium hydroxide (NaOH) aqueous solution was added, and 1,4-butanediol diglycidyl ether (BDDE) was added and mixed in an amount of 100 mol % by weight of hyaluronic acid, so that The total reaction concentration was 10% (w/w) by weight of hyaluronic acid. The mixture contained in said container is placed in a mixer container in which hyaluronic acid and mannitol have been mixed and mixed using a mixer, and then the mixer container is placed in a constant temperature water bath and maintained at 50°C for 2 hours to complete crosslinking. Then, the cross-linked hyaluronic acid hydrogel obtained after the completion of the reaction was roughly cut into a certain size, and a buffer solution (by adding 1.26g/L disodium hydrogen phosphate hydrate (dodecahydrate), 0.46g/L Sodium dihydrogen phosphate hydrate (monohydrate), 7g/L sodium chloride and 3g/L lidocaine hydrochloride were dissolved in a buffer solution prepared in a 500ml bottle container containing water for injection) to wash and swell it for 6 times, 1 hour each time. After pulverizing the hyaluronic acid hydrogel that had been washed and swollen, it was transferred to a 250 ml bottle container and measured for weight, and a buffer solution was added so that the gel weight reached the target weight, thereby performing initial content correction. Upon completion of the initial content correction, the hyaluronic acid hydrogel was squeezed out of the 250 ml bottle container and pulverized using a sieve. The pulverized hyaluronic acid hydrogel was then transferred to a 250ml bottle container and homogenized, then the content was measured and buffer solution was added, thereby making a secondary content correction. The content-corrected hyaluronic acid hydrogel is sterilized by heat treatment at 121° C. or higher for 10 minutes or longer to prepare a cross-linked hyaluronic acid hydrogel according to the present invention.

Example 1-2: According to the present invention using mannitol and cross-linking agent cross-linked cross-linked hyaluronic acid hydrogel Preparation (2)

In order to prepare the cross-linked hyaluronic acid hydrogel cross-linked using mannitol and a cross-linking agent according to the present invention, the following procedure was performed.

Specifically, hyaluronic acid sodium salt with an average molecular weight of 3 million Da, sodium hydroxide, BDDE (1,4-butanediol diglycidyl ether) as a crosslinking agent, and mannitol were weighed out, respectively.

Weigh out 2 g of hyaluronic acid and weigh out 10 mol% of mannitol based on the weight of hyaluronic acid, then place them in a mixer container and mix. To another container (50 mL tube) was added a 0.25 N sodium hydroxide (NaOH) aqueous solution, and 5 mol % of 1,4-butanediol diglycidyl ether (BDDE) based on the weight of hyaluronic acid was added and mixed so that The total reaction concentration was 15% (w/w) by weight of hyaluronic acid. The mixture contained in said container is placed in a mixer container in which hyaluronic acid and mannitol have been mixed and mixed using a mixer, and then the mixer container is placed in a constant temperature water bath and maintained at 30°C for 19 hours to complete crosslinking. Then, the cross-linked hyaluronic acid hydrogel obtained after the completion of the reaction was roughly cut into a certain size, and a buffer solution (by adding 1.26g/L disodium hydrogen phosphate hydrate (dodecahydrate), 0.46g/L Sodium dihydrogen phosphate hydrate (monohydrate), 7g/L sodium chloride and 3g/L lidocaine hydrochloride were dissolved in the buffer solution prepared in the 500ml bottle container containing water for injection) to wash and swell it, 2 hours once and 1 hour twice. After pulverizing the hyaluronic acid hydrogel that had been washed and swollen, it was transferred to a 150 ml bottle container and weighed, and a buffer solution was added so that the gel weight reached the target weight, thereby performing initial content correction. Upon completion of the initial content correction, the hyaluronic acid hydrogel was squeezed out of the 150 ml bottle container and pulverized using a sieve. The pulverized hyaluronic acid hydrogel was then transferred to a 150ml bottle container and homogenized, then the content was measured and a buffer solution was added, thereby making a secondary content correction. The content-corrected hyaluronic acid hydrogel is sterilized by heat treatment at 121° C. or higher for 10 minutes or longer to prepare a cross-linked hyaluronic acid hydrogel according to the present invention.

Example 2: Preparation of cross-linked hyaluronic acid hydrogel cross-linked using sorbitol and cross-linking agent according to the present invention prepare

In order to prepare the cross-linked hyaluronic acid hydrogel cross-linked using sorbitol and a cross-linking agent according to the present invention, the following procedure was performed.

Specifically, hyaluronic acid sodium salt with an average molecular weight of 3 million Da, sodium hydroxide, BDDE (1,4-butanediol diglycidyl ether) and sorbitol as a crosslinking agent were weighed out.

Weigh out 2 g of hyaluronic acid and weigh out 10 mol % of sorbitol based on the weight of hyaluronic acid, then place them in a mixer container and mix. To another container (50 mL tube) was added a 0.25 N sodium hydroxide (NaOH) aqueous solution, and 5 mol % of 1,4-butanediol diglycidyl ether (BDDE) based on the weight of hyaluronic acid was added and mixed so that The total reaction concentration was 15% (w/w) by weight of hyaluronic acid. The mixture contained in said container is placed in a mixer container in which hyaluronic acid and sorbitol have been mixed and mixed using a mixer, and then the mixer container is placed in a constant temperature water bath and maintained at 30°C for 19 hours to complete crosslinking. Then, the cross-linked hyaluronic acid hydrogel obtained after the completion of the reaction was roughly cut into a certain size, and a buffer solution (by adding 1.26g/L disodium hydrogen phosphate hydrate (dodecahydrate), 0.46g/L Sodium dihydrogen phosphate hydrate (monohydrate), 7g/L sodium chloride and 3g/L lidocaine hydrochloride were dissolved in the buffer solution prepared in the 500ml bottle container containing water for injection) to wash and swell it, 2 hours once and 1 hour twice. After pulverizing the hyaluronic acid hydrogel that had been washed and swollen, it was transferred to a 150 ml bottle container and weighed, and a buffer solution was added so that the gel weight reached the target weight, thereby performing initial content correction. Upon completion of the initial content correction, the hyaluronic acid hydrogel was squeezed out of the 150 ml bottle container and pulverized using a sieve. The pulverized hyaluronic acid hydrogel was then transferred to a 150ml bottle container and homogenized, then the content was measured and a buffer solution was added, thereby making a secondary content correction. The content-corrected hyaluronic acid hydrogel is sterilized by heat treatment at 121° C. or higher for 10 minutes or longer to prepare a cross-linked hyaluronic acid hydrogel according to the present invention.

Example 3: Preparation of cross-linked hyaluronic acid hydrogel cross-linked using xylitol and cross-linking agent according to the present invention

In order to prepare the cross-linked hyaluronic acid hydrogel cross-linked using xylitol and a cross-linking agent according to the present invention, the following procedure was performed.

Specifically, hyaluronic acid sodium salt with an average molecular weight of 3 million Da, sodium hydroxide, BDDE (1,4-butanediol diglycidyl ether) and xylitol as a crosslinking agent were weighed out, respectively.

Weigh out 2 g of hyaluronic acid and weigh out 10 mol % xylitol based on the weight of hyaluronic acid, then place them in a mixer container and mix. To another container (50 mL tube) was added a 0.25 N sodium hydroxide (NaOH) aqueous solution, and 5 mol % of 1,4-butanediol diglycidyl ether (BDDE) based on the weight of hyaluronic acid was added and mixed so that The total reaction concentration was 15% (w/w) by weight of hyaluronic acid. The mixture contained in said container is placed in a mixer container in which hyaluronic acid and xylitol have been mixed and mixed using a mixer, and then the mixer container is placed in a constant temperature water bath and maintained at 30° C. for 19 hours to complete crosslinking. Then, the cross-linked hyaluronic acid hydrogel obtained after the completion of the reaction was roughly cut into a certain size, and a buffer solution (by adding 1.26g/L disodium hydrogen phosphate hydrate (dodecahydrate), 0.46g/L Sodium dihydrogen phosphate hydrate (monohydrate), 7g/L sodium chloride and 3g/L lidocaine hydrochloride were dissolved in the buffer solution prepared in the 500ml bottle container containing water for injection) to wash and swell it, 2 hours once and 1 hour twice. After pulverizing the hyaluronic acid hydrogel that had been washed and swollen, it was transferred to a 150 ml bottle container and weighed, and a buffer solution was added so that the gel weight reached the target weight, thereby performing initial content correction. Upon completion of the initial content correction, the hyaluronic acid hydrogel was squeezed out of the 150 ml bottle container and pulverized using a sieve. The pulverized hyaluronic acid hydrogel was then transferred to a 150ml bottle container and homogenized, then the content was measured and a buffer solution was added, thereby making a secondary content correction. The content-corrected hyaluronic acid hydrogel is sterilized by heat treatment at 121° C. or higher for 10 minutes or longer to prepare a cross-linked hyaluronic acid hydrogel according to the present invention.

Comparative Example 1: Preparation of cross-linked hyaluronic acid hydrogel cross-linked using a cross-linking agent according to a conventional method

In order to prepare a crosslinked hyaluronic acid hydrogel crosslinked using a crosslinking agent according to a conventional method, the following procedure was performed.

Specifically, hyaluronic acid sodium salt with an average molecular weight of 3 million Da, sodium hydroxide, and BDDE (1,4-butanediol diglycidyl ether) as a crosslinking agent were weighed out, respectively.

Weigh out 2 g of hyaluronic acid, and add 0.25 N sodium hydroxide (NaOH) aqueous solution to another container (50 mL tube), add 5 mol % of 1,4-butanediol diglycidol based on the weight of hyaluronic acid Ether (BDDE) and mixed so that the total reaction concentration was 15% (w/w) by weight of hyaluronic acid. The mixture contained in the container was placed in the mixer container in which the hyaluronic acid had been mixed and mixed using the mixer, and then the mixer container was placed in a constant temperature water bath and maintained at 30° C. for 19 hours to complete crosslinking. Then, the cross-linked hyaluronic acid hydrogel obtained after the completion of the reaction was roughly cut into a certain size, and a buffer solution (by adding 1.26g/L disodium hydrogen phosphate hydrate (dodecahydrate), 0.46g/L Sodium dihydrogen phosphate hydrate (monohydrate), 7g/L sodium chloride and 3g/L lidocaine hydrochloride were dissolved in the buffer solution prepared in the 500ml bottle container containing water for injection) to wash and swell it, 2 hours once and 1 hour twice. After pulverizing the hyaluronic acid hydrogel that had been washed and swollen, it was transferred to a 150 ml bottle container and weighed, and a buffer solution was added so that the gel weight reached the target weight, thereby performing initial content correction. Upon completion of the initial content correction, the hyaluronic acid hydrogel was squeezed out of the 150 ml bottle container and pulverized using a sieve. The pulverized hyaluronic acid hydrogel was then transferred to a 150ml bottle container and homogenized, then the content was measured and a buffer solution was added, thereby making a secondary content correction. The content-corrected hyaluronic acid hydrogel is sterilized by heat treatment at 121° C. or higher for 10 minutes or longer to prepare a cross-linked hyaluronic acid hydrogel according to the present invention.

Comparative Examples 2, 3, 4, 5, 6 and 7

Commercially available dermal fillers A, B, C, D, E and F were tested as Comparative Examples 2 to 7, respectively.

[Fillers of Comparative Examples 2-7]

A: Juvederm voluma lidocaine, Allergan

B: Restylane lidocaine, Galderma

C: YVOIRE volumes, LG Chem

D: Ellanse, Sinclair

E: Cleviel, Pharma Research Products

F: YVOIRE classic plus, LG Chem

Experimental example 1: Research on the viscoelastic properties of the cross-linked hyaluronic acid hydrogel prepared according to the present invention

In order to study the rheological properties of the prepared Examples 1-2, 2, 3 and Comparative Example 1, they were analyzed using a rheometer. Analysis conditions are as follows.

<analysis conditions>

Analytical conditions for oscillatory and rotational rheometers

In case of complex viscosity (η*) test

(1) Test equipment: Rheometer (Anton Paar Ltd., MCR301)

(2) Frequency: 1Hz

(3) Temperature: 25°C

(4) Strain: 4%

(5) Measurement geometry: 25mm plate

(9) Measuring gap: 1.0mm

The analysis results are shown in Table 1.

【Table 1】

sample Complex viscosity (x10 ⁴ cP) Example 1-2 380 Example 2 381 Example 3 410 Comparative example 1 370

As a result of the complex viscosity analysis of each sample, the complex viscosity of the crosslinked hyaluronic acid hydrogels prepared using mannitol, sorbitol, and xylitol as sugar alcohols was confirmed. The viscosity is higher than that of Comparative Example 1 prepared using only the crosslinking agent.

Experimental Example 2-1: Study on Enzyme Resistance in Vitro (1)

When the cross-linked hyaluronic acid (HA) hydrogel is injected into the body, this cross-linked hyaluronic acid hydrogel undergoes direct decomposition by hyaluronidase attack to be broken down. By injecting hyaluronidase directly into the sample, it is possible to examine the degradation propensity and measure resistance to the enzyme. Specifically, the enzyme resistance test was performed in situ using a rheometer according to the method described below.

After pulverizing the cross-linked hyaluronic acid hydrogel prepared in Examples 1-2, 2 and 3 and the cross-linked hyaluronic acid hydrogel of Comparative Example 1, and then carrying out terminal sterilization, weigh 1 g of sterilized Samples were placed into 50 mL tubes, then 200 uL of hyaluronidase (prepared to 500 units/mL) was added and mixed. The enzyme-mixed sample was loaded onto the rheometer and the temperature was set to 37°C, then the complex viscosity of the sample was measured in real time. The higher the hyaluronidase resistance, the higher the residual rate of the complex viscosity compared to the initial value of the complex viscosity. This means that the higher the residual rate of complex viscosity, the higher the resistance to enzymes. %50Hase (min) was calculated on the basis of the measurement results, and the results are shown in Table 2. %50Hase (min) refers to the time it takes for the complex viscosity of the cross-linked hyaluronic acid hydrogel to decrease to 50% of its initial physical properties due to enzymatic decomposition. In other words, it means that the higher the value of %50Hase(min), the higher the resistance of the cross-linked hyaluronic acid hydrogel.

【Table 2】

sample %50Hase (minutes) Example 1-2 42 Example 2 45 Example 3 41 Comparative example 1 33

As demonstrated in Table 2, the %50Hase (min) of the cross-linked hyaluronic acid hydrogels containing sugar alcohols according to the present invention of Examples 1 to 3 were 42 minutes, 45 minutes and 41 minutes, respectively, while Comparative Example 1 The cross-linked hyaluronic acid hydrogel prepared by the conventional method showed a %50Hase (min) of 33 minutes, thus confirming that the cross-linked hyaluronic acid hydrogel according to the present invention showed relatively high enzyme resistance.

Experimental example 2-2: In vitro enzyme resistance study (2)

Enzyme resistance testing was performed in situ using a rheometer as described below.

After pulverizing the cross-linked hyaluronic acid hydrogel prepared in Example 1-1 and the cross-linked hyaluronic acid hydrogel of Comparative Examples 2 to 4, respectively, and then performing terminal sterilization, 1 g of the sterilized sample was weighed to 50 mL tube, then add 10 uL of hyaluronidase (prepared to 500 units/mL) and mix. The enzyme-mixed sample was loaded onto the rheometer and the temperature was set to 37°C, then the complex viscosity of the sample was measured in real time. %50Hase (min) was calculated on the basis of the measurement results, and the results are shown in FIG. 1 .

As confirmed in Figure 1, the %50Hase (min) of the cross-linked hyaluronic acid hydrogel according to the present invention of Example 1-1 was about 32 minutes, while the cross-linked hyaluronic acid hydrogels prepared by conventional methods of Comparative Examples 2 to 4 Hyaluronic acid hydrogels showed %50Hase(min) of 10, 4 and 6 minutes, respectively, thus confirming that cross-linked hyaluronic acid hydrogels according to the present invention showed 3 times or more times higher enzyme resistance .

Experimental example 3-1: In vitro free radical resistance study (1)

In order to measure the resistance of the cross-linked hyaluronic acid hydrogels prepared in Example 1-1 and the cross-linked hyaluronic acid hydrogels of Comparative Examples 2 to 4 to the degradation induced by peroxyl radicals, the enzyme resistance experiment Also, it was measured in real time using a rheometer by the method described below.

After weighing 1 g of the sterilized sample into a 50 mL tube, 10 uL of 1 molar hydrogen peroxide (H ₂ O ₂ ) and 10 uL of 1 molar ascorbic acid were added and mixed. The mixed sample was loaded onto the rheometer and the temperature was set to 37°C, then the complex viscosity of the sample was measured in real time for 6 hours. % 50H ₂ O ₂ (min) was calculated from the results and shown in FIG. 2 . %50H ₂ O ₂ (min) refers to the time it takes for the complex viscosity of the cross-linked hyaluronic acid hydrogel to decrease to 50% of its initial physical properties (complex viscosity) due to degradation by reactive oxygen species (free radicals). In other words, it means that the higher the value of % 50H ₂ O ₂ (min), the higher the resistance of the cross-linked hyaluronic acid hydrogel to reactive oxygen species (free radicals).

As demonstrated in Figure 2, the % 50H ₂ O ₂ (min) of the crosslinked hyaluronic acid hydrogel according to the invention of Example 1 was about 32 minutes, while the commercially available crosslinked hyaluronic acid hydrogels of Comparative Examples 2 to 4 The hyaluronic acid hydrogels showed % 50H ₂ O ₂ (min) at 5, 7.5 and 18 minutes, respectively, confirming that the cross-linked hyaluronic acid hydrogels according to Example 1-1 of the present invention showed 3-fold or more times higher enzyme resistance.

Experimental example 3-2: In vitro free radical resistance study (2)

In order to measure the resistance of the cross-linked hyaluronic acid hydrogel containing sugar alcohol prepared by Examples 1-2, 2 and 3 and the cross-linked hyaluronic acid hydrogel of Comparative Example 1 to peroxy radical-induced degradation , which was measured in real time using a rheometer by the following method, as in the enzyme resistance experiment.

After weighing 1 g of the sterilized sample into a 50 mL tube, 10 uL of 1 molar hydrogen peroxide (H ₂ O ₂ ) and 10 uL of 1 molar ascorbic acid were added and mixed. The mixed sample was loaded onto the rheometer and the temperature was set to 37°C, then the complex viscosity of the sample was measured in real time for 6 hours. % 50H ₂ O ₂ (min) was calculated from the results and shown in Table 3. %50H ₂ O ₂ (min) refers to the time it takes for the complex viscosity of the cross-linked hyaluronic acid hydrogel to decrease to 50% of its initial physical properties (complex viscosity) due to degradation by reactive oxygen species (free radicals). In other words, it means that the higher the value of % 50H ₂ O ₂ (min), the higher the resistance of the cross-linked hyaluronic acid hydrogel to reactive oxygen species (free radicals).

【table 3】

As demonstrated in Table 3, the %50H ₂ O ₂ (min) of the cross-linked hyaluronic acid hydrogels containing sugar alcohols according to the invention of Examples 1-2, 2 and 3 were 16.5 minutes, 18.4 minutes, respectively and 17.6 minutes, while the cross-linked hyaluronic acid hydrogel prepared by the conventional method of Comparative Example 1 showed %50H ₂ O ₂ (min) of 14.6 minutes, thus confirming that the cross-linked hyaluronic acid hydrogel according to the present invention Gum exhibits a relatively high resistance to free radicals.

Experimental Example 4: In Vitro Heat Resistance Study

The cross-linked hyaluronic acid hydrogel prepared by Example 1-1 and the cross-linked hyaluronic acid hydrogel of Comparative Examples 2 to 4 were subjected to harsh conditions (55°C ± 2°C, 75% RH ± 5% RH ) for 4 hours, and use a rheometer to measure the complex viscosity at 0.02Hz, and calculate the residual rate (%) of the complex viscosity. The results are shown in Figure 3 (unit: cP). The complex viscosity residual ratio (%) represents the ratio of the current complex viscosity to the initial complex viscosity, and it means that the higher the value, the better the stability of physical properties (complex viscosity).

As shown in FIG. 3 , in the cross-linked hyaluronic acid hydrogel according to Example 1-1 of the present invention, about 93% was retained (maintained) compared with the cross-linked hyaluronic acid of Comparative Examples 1 to 3 or higher complex viscosity residual rate, while Comparative Examples 2 to 4 showed low complex viscosity residual rate, so it can be confirmed that the cross-linked hyaluronic acid hydrogel according to the present invention shows excellent stability to heat.

Experimental Example 5: In Vivo Biocompatibility Study

In order to confirm the biocompatibility of the cross-linked hyaluronic acid hydrogel prepared by Example 1-1 and the cross-linked hyaluronic acid hydrogels of Comparative Examples 5 to 7, the tests described below were performed.

Using male rabbits (New Zealand white rabbits, Orient Bio), 0.3 mL samples were subcutaneously administered to 6 rabbits per group, and histopathological examination was performed 4 weeks later. Experiments were performed according to ISO 10993-6 Annex E (Example of Evaluation of Local Biological Effects after Implantation). The results of skin reactions were assessed by the irritation index. The irritation index is an evaluation value of the tissue reaction of the substance when the substance is administered to an animal, and means that the higher the value, the lower the biocompatibility, and no irritation is evaluated as 0.0 to 2.9, and slight irritation is evaluated as 0.0 to 2.9. The evaluation ranged from 3.0 to 8.9, moderate irritation was evaluated from 9.0 to 15.0, and severe irritation was evaluated from >15.0, the results are shown in FIG. 4 . As shown in FIG. 4 , the irritation indices of Comparative Examples 5 and 6 were about 18 and 8, respectively, while the irritation index of Example 1 showed an irritation index value of about 5, so it was comparable to commercially available wrinkle-improving fillers. showed lower or similar levels than , thus confirming that it exhibits suitable biocompatibility as a filler injected into the body.

Claims (23)

1. A crosslinked hyaluronic acid hydrogel, which comprises a crosslinked hyaluronic acid hydrogel,

the crosslinked hyaluronic acid hydrogel comprises hyaluronic acid or a salt thereof, a crosslinking agent and a polyol,

wherein the hyaluronic acid or salt thereof is crosslinked with the crosslinking agent and polyol.

2. The crosslinked hyaluronic acid hydrogel of claim 1, wherein the salt of hyaluronic acid is selected from the group consisting of sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, cobalt hyaluronate, and tetrabutylammonium hyaluronate.

3. The crosslinked hyaluronic acid hydrogel according to claim 1, wherein hyaluronic acid or a salt thereof having an average molecular weight of 1,000,000 to 4,000,000da is crosslinked.

4. The crosslinked hyaluronic acid hydrogel of claim 1, wherein the crosslinking agent is one or more selected from the group consisting of: endogenous polyamines, aldehydes, carbodiimides, divinyl sulfones, butanediol diglycidyl ether (1, 4-butanediol diglycidyl ether: BDDE), ethylene glycol diglycidyl ether (EGDGE), hexanediol diglycidyl ether (1, 6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycidyl ethers, diglycidyl ethers, glycerol polyglycidyl ethers, trimethylpropane polyglycidyl ether, bisoxypropylethylene (1, 2- (bis (2, 3-epoxypropoxy) ethylene), pentaerythritol polyglycidyl ether and sorbitol polyglycidyl ether.

5. The crosslinked hyaluronic acid hydrogel of claim 1, wherein the polyol is a polyol having from 2 to 20 carbon atoms.

6. The crosslinked hyaluronic acid hydrogel according to claim 5, wherein the polyol is selected from one or more of the following: glycerol, 1, 3-propanediol, isopentyl glycol, pentanediol, hexanediol, ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, dipropylene glycol, diglycerol, erythritol, arabitol, adonitol, sorbitol, mannitol, xylitol, dulcitol, glucose, fructose, xylose, trehalose, maltose, sucrose, lactose, and methyl glucoside phosphate.

7. The crosslinked hyaluronic acid hydrogel according to claim 1, having a degree of modification of 0.1 to 100.

8. The crosslinked hyaluronic acid hydrogel of claim 1, comprising from 10mg/g to 35mg/g total hyaluronic acid, based on the total weight of the crosslinked hyaluronic acid hydrogel.

9. A method of preparing the crosslinked hyaluronic acid hydrogel according to any of claims 1-8, the method comprising:

(i) Mixing hyaluronic acid or a salt thereof with a polyol; and

(ii) A mixed solution of a crosslinking agent and an aqueous alkali solution is added to the mixture of hyaluronic acid or a salt thereof and a polyhydric alcohol and reacted.

10. The production method according to claim 9, wherein the concentration of the polyhydric alcohol is 5 to 100mol% based on the hyaluronic acid or a salt thereof.

11. The production method according to claim 9, wherein the concentration of the crosslinking agent is 5 to 100mol% based on the hyaluronic acid or a salt thereof.

12. The production method according to claim 9, wherein the total reaction concentration is 5 to 30% (w/w) based on the weight of the hyaluronic acid.

13. The production method according to claim 9, wherein the concentration of the aqueous alkali solution is 0.1 to 0.5N.

14. The production process according to claim 9, wherein the aqueous alkali solution is NaOH, KOH, naHCO ₃ LiOH, or a combination thereof.

15. A filler comprising the crosslinked hyaluronic acid hydrogel according to claims 1-8.

16. The filler of claim 15, wherein the filler is for dermal injection.

17. The filler according to claim 15, wherein the filler is used for wrinkle improvement, soft tissue repair or volume augmentation or contour correction.

18. The bulking agent of claim 15, further comprising an anesthetic.

19. The filler according to claim 18, wherein the anesthetic is selected from the group consisting of bupivacaine, al Mo Latong, al Mi Luoka, obucaine, benzocaine, betocaine, zhennixirane, bupivacaine, butyl aminobenzoate, bupivacaine, butylaminocaine, ding Yangka factor, cartaine, chloroprocaine, cocoa ethylene, cocaine, cyclomethicaine, dibucaine, quinicaine, dimethcaine, diperoxen, dicycloamine, ecgonine, ethylcloin, etidocaine, beta-eucaine, you Puluo octyl, finamine, formocaine, hexetidine, oxybutycaine isobutyl aminobenzoate, leucaine mesylate, levo Sha Quer, lidocaine, mepivacaine, methyl chloride, miltecaine, naletacaine, ostacaine, oxocaine, hydroxyethocaine, paraethoxycaine, finacaine, phenol, pirocaine, pidocaine, polidocaine, pramoxine, prilocaine, procaine, propamocaine, propisocaine, pseudococaine, pyrrole caine, ropivacaine, salicylalcohol, tetracaine, tol Li Kayin, mepivacaine, zolamine and salts thereof.

20. The bulking agent of claim 15, further comprising a buffer solution and an isotonic agent.

21. A method of preparing the filler of claim 15, the method comprising the steps of:

(a) Mixing hyaluronic acid or a salt thereof with a polyol;

(b) Adding a mixed solution of a crosslinking agent and an aqueous alkali solution to the mixture of hyaluronic acid or a salt thereof and a polyhydric alcohol and reacting them to prepare a crosslinked hyaluronic acid hydrogel;

(c) Performing rough cutting on the hyaluronic acid hydrogel prepared in (b);

(d) Washing and swelling the rough cut hyaluronic acid hydrogel prepared in (b) using a buffer solution; and

(e) Pulverizing the hyaluronic acid hydrogel washed and swelled in (d).

22. A prefilled syringe filled with the filler according to claim 15.

23. A method for wrinkle improvement, soft tissue repair or volume augmentation or contour correction, the method comprising injecting the filler of claim 15.